Analytical Chemistry Chemical Cytometry Quantitates Superoxide

Switzerland), was placed inside the chromatographic oven to
bypass the combustion furnace when required. The valve has an
extension between the body and the actuator which allows the
valve to be mounted in the heated zone while the handle remains
outside at ambient temperature. This valve prevented the solvent
from entering the combustion unit and therefore enlarged the
catalytic activity of the Cu and Pt wires. Additionally, the valve
allowed the direct connection of the column with the EI source
for conventional GC-MS work in order to identify the different
species under analysis by their fragmentation pattern. Moreover,
such qualitative analysis is essential to assess peak purity for each
compound. For postcolumn isotope dilution analysis the valve was
initially in the “load” position (combustion oven bypassed) and
the valve was manually switched to the “inject” position at the
same time that the EI filament switched on. All connections
between the valve and the rest of the components of the system
were performed by means of 0.32 mm i.d. deactivated fused silica
capillaries and fixed to the valve with appropriate polyimide coated
fused silica adapters (VICI AG International, Schenkon, Switzerland).
Combustion Furnace. The laboratory-made combustion furnace
consisted of a 60 cm long ceramic tube (3 mm O.D., 0.5 mm I.D.)
(Elemental microanalysis, Devon, U. K.) filled with copper and
platinum wires and heated by a Nichrome wire. Proper thermal
isolation of the combustion furnace was provided with glass wool.
The combustion furnace was set vertically on top of the GC with
the lower end of the ceramic tube inside the chromatographic
oven to avoid cold spots after the separation. High temperatures
were accurately controlled using a temperature sensor and an
external controller, allowing temperature settings inside the tube
to be within ±1 °C over a wide temperature range (50-1200 °C).
The copper wires were previously oxidized by passing an oxygen
flow (1 mL/min) at 450 °C during 4-5 h and this procedure was
performed on a weekly basis to preserve its oxidizing capabilities.
Fused silica capillaries (0,32 mm i.d.) were used to connect the
furnace to the valve and to the ion source respectively. For this
purpose a length (∼1 cm) at one end of each capillary was
uncoated, to prevent the polyimide coating to be burnt, and
introduced into the ceramic tube. Reducing unions 1/8′′ to 1/16′′
(SGE, Victoria, Australia) and appropriate graphite ferrules (0.5
mm i.d.) were used to hold the capillaries in place at both sides
of the ceramic tube. For oxidation and combustion, the furnace
was operated at 850 °C.
Gas Cylinder and Mass Flow Controller. The 13 CO2 container
was a dual inlet 5 L high pressure stainless steel gas cylinder
(Iberfluid, Barcelona, Spain). The cylinder was equipped on
one end with, in this order, an opening valve, a Swagelok tee
where helium could be introduced from a high pressure
cylinder, and a second opening valve connected to the other
end of the tee. This second valve was connected to a septum
for the manual injection of gases into the cylinder. Before the
cylinder was pressurized, 13 CO2 was injected into the container
by means of a gastight syringe (Hamilton, Reno, U. S. A.)
through this valve. After the injection of the tracer, the valve
was closed and the container was pressurized up to 6 bar with
helium using the connection in the Swagelok tee. When the
set pressure was reached the filling valve was also closed. At
the other end of the cylinder a pressure gauge, an opening
6864 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010
valve and a mass-flow controller (Bronkhorst, Ruurlo, Netherlands)
calibrated for He were coupled for the accurate control
of the tracer flow. The opening valve was closed during the
filling of the cylinder, remaining open the rest of the time. The
flow rate for the postcolumn spike was set at 0.5 mL/min.
Effluent and spike flows were mixed after combustion by means
of a 0.25 mm bore stainless steel microvolume “Y” connector
(VICI AG International, Schenkon, Switzerland).
The whole instrumental setup is shown in Figure 1. As can be
observed, the instrument can be operated in the standard GC-
MS configuration (qualitative) or in the combustion-postcolumn
configuration (quantitative) depending on the position of the
switching valve.
Procedures. Preparation of 13 CO2. The spike was prepared
from 13 C enriched Na2CO3 (99%). An accurate weighed amount
(∼200 mg) was placed in a 25 mL three-necked round-bottomed
flask, previously purged with He to avoid natural abundances
CO2 contamination from ambient air. A small quantity (300 µL)
of concentrated H3PO4, was injected into the flask through a
septum cap. After the acid-base reaction, 4 mL of the gaseous
phase, containing 13 CO2 diluted in He, were removed using a
gastight syringe and injected into the container shown in
Figure 1.
Calibration of the 13 CO2 Postcolumn Flow. The flow rate of
the spike could be accurately controlled by the mass flow
controller between 0.1 and 5 mL min -1 . In our experiments, it
was set at 0.5 mL min -1 . The exact mass flow (ng of 13 CO2 per
min) being mixed with the natural CO2 coming from the
column and combustion furnace was determined by adding
internal standards of known concentration spiked to the sample
as described before. 11,15
Quantification using Postcolumn Isotope Dilution. In our case,
the isotope ratio 12 C/ 13 C was measured as the signal ratio at
masses 44 and 45 (I44/I45) corresponding to the continuous
blend of natural abundance 12 CO2 present in the chromatographic
eluent and the enriched 13 CO2, added postcolumn.
Selected Ion Monitoring (SIM) at masses 44.0 and 45.0 was
performed for the duration of the chromatogram with 70 ms
integration time per mass. The mass window was ∼0.1 mass
units. Then, the isotope ratio in the blend, Rb ) I44/I45, was
calculated to build the isotope ratio chromatogram (Rb vs time).
The postcolumn isotope dilution equation, 7 shown as equation 1
below, was then applied to every point in the chromatogram
to obtain the mass-flow chromatogram (ng of C/min vs time).
The integration of the mass flow chromatogram directly
provided the amount of carbon (in ng) eluted in each chromatographic
peak.
MF n ) MF t
AW n
13
At AWt An 12( Rb - Rt 1 - RbRn) In this equation MFn corresponds to the mass flow of carbon
from the natural abundance sample injected, whereas MFt
corresponds to the mass flow of carbon from the postcolumn
spike or tracer. AWn and AWt correspond to the atomic weight
of carbon in the sample and tracer, respectively. The isotope
abundances At 13 and An 12 correspond to the isotopic composition
of 13 C in the tracer and 12 C in the sample. Finally, Rt is the
(1)